Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Oct 28;20(21):5348.
doi: 10.3390/ijms20215348.

Immunomodulatory Protein from Nectria haematococca Induces Apoptosis in Lung Cancer Cells via the P53 Pathway

Jing-Jing Wang  1 , Yan Wang  2 , Lizhen Hou  3 , Fengjiao Xin  4 , Bei Fan  5 , Cong Lu  6 , Lijing Zhang  7 , Fengzhong Wang  8 , Shuying Li  9
Affiliations

Immunomodulatory Protein from Nectria haematococca Induces Apoptosis in Lung Cancer Cells via the P53 Pathway

Jing-Jing Wang et al. Int J Mol Sci. .

Abstract

Our previous research has shown that a fungal immunomodulatory protein from Nectria haematococca (FIP-nha) possesses a wide spectrum of anti-tumor activities, and FIP-nha induced A549 apoptosis by negatively regulating the PI3K/Akt signaling pathway based on comparative quantitative proteomics. This study further confirmed that the anti-lung cancer activity of FIP-nha was significantly stronger than that of the reported LZ-8 and FIP-fve. Subsequently, 1H NMR-based metabolomics was applied to comprehensively investigate the underlying mechanism, and a clear separation of FIP-nha-treated and untreated groups was achieved using pattern recognition analysis. Four potential pathways associated with the anti-tumor effect of FIP-nha on A549 cells were identified, and these were mainly involved in glycolysis, taurine and hypotaurine metabolism, fructose and mannose metabolism, and glycerolipid metabolism. Metabolic pathway analysis demonstrated that FIP-nha could induce A549 cell apoptosis partly by regulating the p53 inhibition pathway, which then disrupted the Warburg effect, as well as through other metabolic pathways. Using RT-PCR analysis, FIP-nha-induced apoptosis was confirmed to occur through upregulation of p53 expression. This work highlights the possible use of FIP-nha as a therapeutic adjuvant for lung cancer treatment.

Keywords: A549; NMR metabolomics; Nectria haematococca; fungal immunomodulatory protein.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Cytotoxicity assay of recombinant fungal immunomodulatory protein from Nectria haematococca (rFIP-nha) in A549 cells. (A) Morphological observation of A549 cells after rFIP-nha treatment. A549 cells were treated with 0, 4, 6, 8, 16, or 32 μg/mL rFIP-nha for 24 h, and micrographs were collected on a Zeiss microscope at 200 × magnification. (B) Cell viability assay. A549 cells and normal HEK293 cells were treated with varying concentrations (0, 2, 4, 8, 16, 32, or 64 μg/mL) of rFIP-nha for 24 h, followed by CCK assays. (C) Comparison of cytotoxicity between rFIPs. A549 cells were treated with varying concentrations (0, 8, 16, or 32 μg/mL) of rFIP-nha, rLZ-8, or rFIP-fve for 24 h, followed by CCK assays to estimate cell viability. Each bar represents mean ± SD (n = 3). **: p < 0.01 compared to control (0 μg/mL). +: p < 0.05 between different FIPs at the same concentration.
Figure 2
Figure 2
Typical 1H NMR spectra (δ0.5-9.0) of A549 cell extracts obtained from groups treated with (S) and without (C) rFIP-nha. The region of δ6.0–9.5 (in the dashed box) was magnified 20 times compared with the corresponding region of δ0.5–6.0 for clarity. Keys: 1-MH, 1-methylhistideine; 3-HB, 3-hydroxybutyrate; AA, acetoacetate; Ace, acetate; Act, acetone; Ala, alanine; Bu, butyrate; Cho, choline; Cit, citrate; Cn, creatinine; Cr, creatine; EA, ethanolamine; Eth, ethanol; For, formate; G, glycerol; Glc, glucose; Gln, glutamine; His, histidine; HIV, 3-hydroxyisovalerate; HOD, the residual water signals; IB, isobutyrate; Ile, isoleucine; KIV, 2-ketoisovalerate; Lac, lactate; Lys, lysine; Mal, mannitol; MG, 7-methylguanosine; MNA, 1-methylnicotinamide; Mol, methanol; p-AB, p-aminobenzoate; PC, phosphocholine: Phe, phenylalanine; Py, pyruvate; SCFA, short-chain fatty acid; Tau, Taurine; Thr, threonine; TMA, trimethylamine; TMAO, trimethylamine N-oxide; Tyr, tyrosine; Val, valine.
Figure 3
Figure 3
(A) 2D principal component analysis (PCA) scores plots based on 1H NMR spectra of A549 cells obtained from groups C and S. (B) Partial least squares-discriminant analysis (PLS-DA) scores plots based on 1H NMR spectra of A549 cells obtained from groups C and S. The experimental group of A549 cells (group S) were treated with 16 μg/mL FIP-nha for 24 h, and the control group (group C) was treated with same volume of PBS for 24 h.
Figure 4
Figure 4
Orthogonal projection to latent structure with discriminant analysis (OPLS-DA) scores plots (A) derived from 1H NMR spectra of A549 cells. Corresponding coefficient loading plots (B) obtained from groups C and S and cross validation (C) by permutation test (n = 200). The significance of metabolites variations between the two classes is shown in a color map. Peaks in the negative direction demonstrate metabolites of group C are more abundant. In the group S, if metabolites are more abundant, the peaks will in the positive direction. Figure 2 shows the keys of the assignment.
Figure 5
Figure 5
Summary of altered metabolic pathways and regulatory mechanism of rFIP-nha against A549 cells. HK, hexokinase; PGI, phosphoglucose isomerase; PFK1, phosphofructokinase 1; Aldo, aldolase; TPI, triose phosphate isomerase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; Eno, enolase; PK, pyruvate kinase; LDH, lactate dehydrogenase; TCA, tricarboxylic acid cycle; G6PD, glucose-6-phosphate dehydrogenase; TPA, taurine-pyruvate aminotransferase; pfp, pyrophosphate-fructose-6-phosphate 1-phosphotransferase; mtlD, mannitol-1-phosphate 5-dehydrogenase; M1Pase, mannitol-1-phosphatase; gldA, glycerol dehydrogenase; DAK, dihydroxyacetone kinase; GLUTs, glucose transporters; HER2, receptor tyrosine-protein kinase erbB-2; PI3K, phosphatidylinositol 3-kinase; Akt, serine-threonine kinase; mTOR, mammalian target of rapamycin; HIF-1α, hypoxia-inducible factor 1α; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; c-Myc, a transcription factor; p53, a tumor suppressor; Pten, a human tumor suppressor gene on chromosome 10; AMPK, AMP-activated protein kinase; TSC2, tuberous sclerosis complex 2; TIGAR, TP53-induced glycolysis and apoptosis regulator; GLS2, glutaminase 2; SCO2, cytochrome c oxidase 2; COX4, cytochrome c oxidase subunit 4. HER2-mediated PI3K-Akt-mTOR signaling plays a pivotal role in promoting glycolysis in tumor cells via the activation of HIF-1α, NF-κB, and c-Myc. p53 plays a key role in the process of suppressing glycolysis and promoting oxidative phosphorylation (OXPHOS) by interacting with various enzymes and other molecules, including SCO2, TIGAR, GLUT1, GLUT4, GLS2, and PGM. Different metabolic pathways represented within the dashed box. The arrow represents stimulatory modification, the T-shaped arrow represents inhibitory modification.
Figure 6
Figure 6
Analysis of differential p53 expression in A549 cells after rFIP-nha treatment. A549 cells were treated with 16 μg/mL of rFIP-nha for 24 h, followed by RT-PCR assays to estimate p53 expression changes. Each bar represents mean ± SD (n = 3); *: p < 0.05 compared to control (0 μg/mL).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Chen W., Zheng R., Baade P.D., Zhang S., Zeng H., Bray F.I., Jemal A., Yu X.Q., He J. Cancer statistics in China, 2015. CA Cancer J. Clin. 2016;66:115–132. doi: 10.3322/caac.21338. - DOI - PubMed
    1. Chen Y., Ma Z., Li A., Li H., Wang B., Zhong J., Min L., Dai L. Metabolomic profiling of human serum in lung cancer patients using liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry and gas chromatography/mass spectrometry. J. Cancer Res. Clin. Oncol. 2015;141:705–718. doi: 10.1007/s00432-014-1846-5. - DOI - PubMed
    1. Baird R.D., Kaye S.B. Drug resistance reversal—Are we getting closer? Eur. J. Cancer. 2003;39:2450–2461. doi: 10.1016/S0959-8049(03)00619-1. - DOI - PubMed
    1. Kino K., Yamashita A., Yamaoka K., Watanabe J., Tanaka S., Ko K., Shimizu K., Tsunoo H. Isolation and characterization of a new immunomodulatory protein, ling zhi-8 (LZ-8), from Ganoderma lucidium. J. Biol. Chem. 1989;264:472–478. - PubMed
    1. Li J., Cheng C., Yang W., Yang C., Ou Y., Wu M., Ko J. FIP-gts Potentiate Autophagic Cell Death Against Cisplatin-resistant Urothelial Cancer Cells. Anticancer Res. 2014;34:2973–2983. - PubMed

MeSH terms